Severe acute respiratory syndrome (SARS) is a recently-recognized, febrile severe lower respiratory illness that is the result of an infection caused by a novel coronavirus (SARS-CoV) (1-5). The global outbreak of SARS was contained, but concerns remain over the possibility of future recurrences, especially with recent reports of laboratory-acquired infections (6). However, no effective treatment or prophylaxis is currently available to combat this deadly virus (7, 8).
Like other coronaviruses, SARS-CoV is an enveloped virus containing a large, positive-stranded RNA genome that encodes viral replicase proteins and structural proteins including spike (S), membrane (M), envelope (E), nucleocapsid (N), and several uncharacterized proteins (4, 5, 9). Phylogenetic analyses indicate that SARS-CoV is distinct from the three known antigenic groups of coronaviruses. Therefore, post-genomic characterization of SARS-CoV is important for developing anti-SARS therapeutics and vaccines (10, 11).
Coronavirus infection is initiated by attachment of the S protein to the specific host receptor, which triggers a conformational change in the S protein. The S protein of SARS-CoV is a type I transmembrane glycoprotein with a predicted length of 1,255 amino acids that contains a leader (residues 1-14), an ectodomain (residues 15-1190), a transmembrane domain (residues 1191-1227), and a short intracellular tail (residues 1227-1255) (5). Unlike many other coronaviruses, such as the mouse hepatitis virus (MHV)(12, 13), in which the S protein is post-translationally cleaved into S1 and S2 subunits, no typical cleavage motif has been identified in the SARS-CoV S protein (5). Nonetheless, its S1 and S2 domains were predicted by sequence alignment with other coronavirus S proteins (5, 14). The S2 domain (residues 681-1255) of SARS-CoV S protein containing a putative fusion peptide and two heptad repeat (HR1 and HR2) regions is responsible for fusion between viral and target cell membranes. It has been found that the HR1 and HR2 regions can associate to form a six-helix bundle structure (15-18), resembling the fusion-active core of the HIV gp41 (19) and the MHV S protein (20, 21). The S1 domain of SARS-CoV S protein mediates virus-binding with angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV on susceptible cells (22-25). Recently, a 193-amino-acid small fragment within S1 domain (residues 318-510) was identified as a receptor-binding domain (RBD), which is sufficient to associate with ACE2 (26-28).
The S proteins of coronaviruses are major antigenic determinants that induce the production of neutralizing antibodies (29, 30). Thus, it logically follows to use S protein as an antigen for vaccine development (30). Recently, it has been shown that the S protein of SARS-CoV is a major inducer of protective immunity among structural proteins (31). Yang, et al. (32) reported that a DNA vaccine candidate encoding the S protein induced SARS-CoV neutralization (neutralizing antibody titers ranged from 1:25 to 1:150) and protective immunity in mice, and it was proven that the protection was mediated by neutralizing antibodies but not by a T-cell-dependent mechanism. Bisht, et al. (33) demonstrated that the S protein of SARS-CoV expressed by attenuated vaccinia virus (MVA) elicited S-specific antibodies with SARS-CoV-neutralizing antibody titer of 1:284, and protectively-immunized mice against SARS-CoV infection as shown by reduced titers of SARS-CoV in the respiratory tracts of mice after the challenge infection. Bukreyev, et al. (34) reported that mucosal immunization of African green monkeys with an attenuated parainfluenza virus (BHPIV3) expressing the SARS-CoV S protein induced neutralizing antibodies with neutralization titers ranging from 1:8 to 1:16 and protected animals against the challenge infection. These data indicate that the S protein of SARS-CoV is a protective antigen capable of inducing neutralizing antibodies, although its antigenic determinants remain to be defined.
We have recently demonstrated that the receptor-binding domain (RBD) of SARS-CoV S protein is a major target of neutralizing antibodies induced in patients infected with SARS-CoV and in animals immunized with inactivated viruses or S proteins (35, 36). Therefore, we used the recombinant RBD of the SARS-CoV S protein as an immunogen to induce neutralizing monoclonal antibodies (mAbs).